AbstractCobia (Rachycentron canadum) is a coastal pelagic migratory fish species of tropical and subtropical waters, where it is an important game fish and it has been commercially expanded in offshore aquaculture systems. Understanding population connectivity is of utmost importance to the sustainable use and conservation of aquatic resources, and information on genetic diversity and structure is key element in unraveling differentiation when no clear physical barriers exist. In the present study, cobia genetic diversity and structure were depicted using mitochondrial DNA cytochrome b sequencing and microsatellite genotyping in samples from the Southwestern Atlantic and showed that a major single population inhabits the southern hemisphere. Cytochrome b sequencing also suggested that the Indian Ocean is the center of origin for this species' diversification. A hierarchical analysis of AMOVA compared sampling locations from the Northwestern Atlantic (from a previous study) with the Southwestern ones using nine shared microsatellite markers. Differentiation among groups (FCT = 0.41), Bayesian clustering analysis, and complementary ordination analyses (by discriminant analysis of principal components [DAPC] and factorial correspondence analysis [3D‐FCA]) presented a clear separation between the two hemispheres, supported by a Lagrangian model that explained the ocean dynamics over larval retention on the Western Atlantic. Another genetic subgroup intermingled with the main Southwestern group may also exist further south, probably associated with the Vitória‐Trindade Ridge and the local current systems. The distribution of this species in metapopulations is of extreme relevance for fisheries and fish hatcheries management in the Atlantic Ocean.